RESEARCH
Evidence for
Elizabethkingia anophelis
Transmission from Mother
to Infant, Hong Kong
Susanna K.P. Lau,1 Alan K.L. Wu,1 Jade L.L. Teng,1 Herman Tse,1 Shirly O.T. Curreem,
Stephen K.W. Tsui, Yi Huang, Jonathan H.K. Chen, Rodney A. Lee, Kwok-Yung Yuen, Patrick C.Y. Woo
Elizabethkingia anophelis, recently discovered from mos-
quito gut, is an emerging bacterium associated with neo-
natal meningitis and nosocomial outbreaks. However,
its transmission route remains unknown. We use rapid
genome sequencing to investigate 3 cases of E. anoph-
elis sepsis involving 2 neonates who had meningitis and 1
neonate’s mother who had chorioamnionitis. Comparative
genomics revealed evidence for perinatal vertical transmis-
sion from a mother to her neonate; the 2 isolates from these
patients, HKU37 and HKU38, shared essentially identical
genome sequences. In contrast, the strain from another
neonate (HKU36) was genetically divergent, showing only
78.6% genome sequence identity to HKU37 and HKU38,
thus excluding a clonal outbreak. Comparison to genomes
from mosquito strains revealed potential metabolic adapta-
tions in E. anophelis under different environments. Mater-
nal infection, not mosquitoes, is most likely the source of
neonatal E. anophelis infections. Our findings highlight the
power of genome sequencing in gaining rapid insights on
transmission and pathogenesis of emerging pathogens.
M icrobial genome sequencing can enhance diagnosis
and control of infectious diseases (1,2). Its ultimate
molecular resolution is superior to other phenotypic and
genotypic tests and enables not only rapid microbial iden-
tification but also characterization of transmission events.
The technique has been applied in large-scale infectious
disease outbreaks such as those caused by Escherichia coli
O104:H4, Staphylococcus aureus, Streptococcus pyogenes,
Enterococcus faecium, Pseudomonas aeruginosa, Vibrio
Author affiliations: The University of Hong Kong, Hong Kong
(S.K.P. Lau, J.L.L. Teng, H. Tse, S.O.T. Curreem, Y. Huang,
J.H.K. Chen, K.-Y. Yuen, P.C.Y. Woo); State Key Laboratory of
Emerging Infectious Diseases, Research Centre of Infection and
Immunology, Carol Yu Centre for Infection, Hong Kong (S.K.P.
Lau, H. Tse, K.Y. Yuen, P.C.Y. Woo); Pamela Youde Nethersole
Eastern Hospital, Hong Kong (A.K.L. Wu, R.A. Lee); School of
Biomedical Sciences, The Chinese University of Hong Kong,
Hong Kong (S.K.W. Tsui)
DOI: http://dx.doi.org/10.3201/eid2102.140623
232 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 2, February 2015
cholerae, and mycobacteria (3–14). However, the routine
application of this method in diagnostic microbiology and
infection control, especially for less well-defined, emerging
pathogens, is yet to be explored.
Elizabethkingia anophelis is a recently discovered bac-
terium isolated from the midgut of the Anopheles gambiae
mosquito in 2011 (15). The genus Elizabethkingia also in-
cludes E. meningoseptica (previously named Chryseobac-
terium/Flavobacterium meningosepticum) and E. miricola
(16). E. meningoseptica causes neonatal sepsis and infec-
tions in immunocompromised persons. E. anophelis has
also recently been reported to cause neonatal meningitis in
the Central African Republic, and a nosocomial outbreak
was reported in an intensive care unit in Singapore (17–19).
However, the role of mosquitoes or other sources in the
transmission of E. anophelis remains unclear.
In 2012, we encountered 3 cases of Elizabethkingia
sepsis associated with meningitis in 2 neonates and cho-
rioamnionitis in a neonate’s mother in a hospital in Hong
Kong. Three strains of Elizabethkingia-like, gram-neg-
ative bacilli sharing similar phenotypic characteristics
were isolated from the 3 patients, but confident identifi-
cation results were not obtained by matrix-assisted laser
desorption ionization/time-of-flight (MALDI-TOF) mass
spectrometry and 16S rRNA gene sequencing. Moreover,
clinical and microbiological data did not provide adequate
clues about the possible transmission route. We therefore
attempted to use draft genome sequencing to rapidly dis-
sect transmission pathways and confirm the identity of
the species.
Materials and Methods
Setting and Patients
The 3 patients were hospitalized in an acute regional hos-
pital, Pamela Youde Nethersole Eastern Hospital, which
is situated in the eastern area of Hong Kong Island. This
study was approved by the Institute Review Board, Hos-
pital Authority, Hong Kong (reference HKEC-2013-051).
1
These authors contributed equally to this article.

Microbiological Methods
Bacterial cultures and phenotypic identification were per-
formed according to standard protocols by using the Vitek
II system (bioMérieux, Marcy l’Etoile, France). Antimi-
crobial drug susceptibility testing was performed by E-test
method for vancomycin and Kirby-Bauer disk diffusion for
other drugs; because interpretative criteria for Elizabeth-
kingia were lacking, results were interpreted according to
Clinical and Laboratory Standards Institute for Pseudomo-
nas aeruginosa (20). MALDI-TOF mass spectrometry was
performed by the direct transfer method as described previ-
ously (21), with modifications by using the Bruker Dalton-
ics microflex LT system with Reference Library Biotyper
version 3.1 (Bruker Daltonik GmbH, Leipzig, Germany).
Full 16S rRNA gene amplification and squencing were
performed according to previously published protocols
with modifications (22,23). Pulsed-field gel electrophoresis
(PFGE) was performed by using the CHEF Mapper XA
system (Bio-Rad, Hercules, CA, USA) and restriction en-
donuclease XbaI as described previously (8,22).
Draft Genome Sequencing and Analysis
The draft genome sequences of the 3 E. anophelis strains
were determined by high-throughput sequencing with the
Illumina HiSeq 2500 system (Illumina, San Diego, CA,
USA). Samples of 50 ng of genomic DNA were extract-
ed by using a genomic DNA purification kit (QIAGEN,
Hilden, Germany) from cultures grown overnight on blood
agar at 37°C, as described previously (24,25). Each sample
was sequenced by 151-bp paired-end reads with mean li-
brary size of 350 bp. Sequencing errors were corrected by
k-mer frequency spectrum analysis using SOAPec (http://
soap.genomics.org.cn/about.html). De novo assembly was
performed in SOAPdenovo2 (http://soap.genomics.org.cn/
soapdenovo.html). Prediction of protein coding regions
and automatic functional annotation was performed by
using Glimmer3 (26) and the RAST (Rapid Annotations
using Subsystem Technology) server (27). Antibiotic re-
sistomes were identified by using the Antibiotic Resistance
Genes Database (28). BLASTn comparisons were run in
BLAST+ (http://blast.ncbi.nlm.nih.gov/Blast.cgi) with an
E-value cutoff of 10.0. In addition, manual annotation was
performed on putative virulence and antibiotic resistance
genes by protein domain predictions and multiple sequence
alignments with orthologous genes. Intergenomic distance
was calculated by using Genome-to-Genome Distance Cal-
culator 2.0 (http://ggdc.dsmz.de/distcalc2.php) (29).
Results
Patients
In July 2012, a 21-day-old male neonate (patient 1) was
admitted to Pamela Youde Nethersole Eastern Hospital for
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 2, February 2015 233
E. anophelis Transmission from Mother to Infant
fever of 1 day’s duration. He was born at the same hospital
21 days earlier at 41 weeks’ gestation by vaginal delivery
and was discharged on day 3. Physical examination did not
show obvious infective focus. Serum C-reactive protein
(CRP) was elevated to 109 mg/L. Lumbar puncture was
performed; analysis of cerebrospinal fluid (CSF) showed
polymorph pleocytosis, elevated protein levels, and low
glucose levels (Table). Treatment was initiated for bacte-
rial meningitis with empirical intravenous ampicillin and
cefotaxime. Blood and CSF cultures recovered a gram-
negative bacillus, designated HKU36. Antimicrobial drugs
were changed to vancomycin, piperacillin, and rifampin on
day 3. The patient was discharged after 3 weeks of intrave-
nous drug treatment, without neurologic sequelae (Figure
1). The neonate’s mother was admitted to the same hospi-
tal 1 day after the infant’s admission for postpartum fever,
chills, rigor, and abdominal pain. Transvaginal ultrasound
showed no retained gestational products. Serum CRP level
was elevated to 109 mg/L; however, blood cultures were
negative. She was treated with intravenous cefuroxime and
metronidazole and discharged on day 6 with oral cefurox-
ime and metronidazole.
In November 2012, a 33-year-old woman in week 30
of pregnancy (patient 2) was admitted to the same hospi-
tal because of prolonged premature rupture of membranes.
She stayed at the same antenatal ward and in the same cu-
bicle as the mother of patient 1 (Figure 1). Fever devel-
oped in the patient 3 days after admission, and clinical tests
showed peripheral leukocytosis with neutrophilia (Table).
Serum CRP was elevated to 108 mg/L. Treatment with in-
travenous penicillin G was commenced, and an emergency
lower segment cesarean section was performed. Placental
and uterine swab cultures recovered a gram-negative bacil-
lus, designated HKU37. Blood cultures were negative. An-
timicrobial drug treatment was changed to cefuroxime and
metronidazole, followed by oral ciprofloxacin for 1 week.
Her fever subsided, and she was discharged on day 8.
The baby girl (patient 3) of patient 2 was pale and
flaccid at birth; apnea of prematurity developed, requir-
ing cardiopulmonary resuscitation. Peripheral leukopenia
and metabolic acidosis were also detected, and serum CRP
level was elevated to 70.6 mg/L. Chest radiograph showed
bilateral ground-glass appearance. Lumbar puncture was
performed, and CSF showed lymphocytic pleocytosis with
elevated protein levels and low glucose levels (Table). Ul-
trasound of the brain showed grade I to II intraventricular
hemorrhages. Empirical intravenous ampicillin and ce-
fotaxime at meningitic dose was started. Blood and CSF
cultures recovered a gram-negative bacillus, designated
HKU38. Antimicrobial drug therapy was changed to intra-
venous vancomycin, piperacillin/tazobactam, and rifampin
on day 3, continuing for 3 weeks. Necrotizing enterocolitis
and neonatal jaundice developed, but both resolved with
RESEARCH

Table. Clinical characteristics and results of testing for 3 patients infected with Elizabethkingia anophelis, Hong Kong, 2012*
Characteristics Patient 1 Patient 2† Patient 3
Patient age/sex 21 d/M 33 y/F 0 d/F
Signs/symptoms Fever Fever, PPROM Apnea at birth
Blood test results
Total leukocytes, × 109 cells/L 16.0 (5.0–19.5) 15.2 (3.7–9.3) 5.1 (10.0–27.0)
Neutrophils, × 109 cells/L 6.8 (2.0–9.5) 12.5 (1.8–6.2) 1.2 (5.0–17.0)
Lymphocytes, × 109 cells/L 6.8 (2.5–11.0) 1.7 (1.0–3.2) 3.4 (3.0–10.0)
Monocytes, × 10 cells/L
9
2.3 (0.2–1.2) 0.8 (0.2–0.7) 0 (0.5–2.0)
Hemoglobin, g/dL 14.0 (11.0–19.0) 10.7 (11.5–15.4) 16.1 (13.5–19.5)
Platelets, × 109/L 180 (180–460) 241 (160–420) 186 (100–300)
C-reactive protein, mg/L 109 (<8.0) 108 (<5.0) 70.6 (<8.0)
CSF test results
Total leukocytes, × 106 cells/L 1,445 NA 5,850
Polymorphs, % 67 NA 1
Lymphocytes, % 33 NA 99
Protein, g/L 1.33 (0.15–0.45) NA 2.69 (0.15–0.45)
Glucose, mmol/L 2.2 (2.8–4.4) NA 1.5 (2.8–4.4)
CSF/serum glucose, % 38 NA 24
Positive culture sites for E. anophelis Blood, CSF Placental swab, uterine swab Blood, CSF
Phenotypic characteristics of isolates
Colony pigment Pale yellow None None
Citrate utilization Negative Delayed positive Delayed positive
Antimicrobial drug susceptibilities of isolates
Ampicillin Resistant Resistant Resistant
Pipercillin Susceptible Susceptible Susceptible
Cefoperazone/sulbactam Susceptible Susceptible Susceptible
Cefotaxime Intermediate Resistant Resistant
Ceftazidime Resistant Resistant Resistant
Imipenem Resistant Resistant Resistant
Amikacin Resistant Resistant Resistant
Gentamicin Resistant Resistant Resistant
Kanamycin Resistant Resistant Resistant
Streptomycin Resistant Resistant Resistant
Tobramycin Resistant Resistant Resistant
Ciprofloxacin Susceptible Susceptible Susceptible
Moxifloxacin Susceptible Susceptible Susceptible
Tetracycline Resistant Resistant Resistant
Trimethoprim/sulfamethoxazole Susceptible Susceptible Susceptible
Rifampin Susceptible Susceptible Susceptible
Chloramphenicol Resistant Resistant Resistant
Vancomycin MIC, μg/mL 16 4 4
Antimicrobial drug regimen Ampicillin + cefotaxime; Penicillin G; cefuroxime + Ampicillin + cefotaxime;
vancomycin + piperacillin + metronidazole; ciprofloxacin vancomycin +
rifampin pipercillin/tazobactam +
rifampin
Complications None None Respiratory distress,
intraventricular
hemorrhage
*Reference ranges are shown in parentheses. PPROM, prolonged premature rupture of membranes; CSF, cerebrospinal fluid.
†Mother of patient 3.

treatment (Figure 1). The infant was discharged on day 54
without neurologic sequelae.
Clinical and Microbiological Investigations
The 3 isolates from these patients, HKU36–38, were non-
motile, oxidase-positive, non–glucose-fermenting, gram-
negative bacilli. Their phenotypic characteristics are
summarized in the Table and online Technical Appendix
Table 1 (http://wwwnc.cdc.gov/EID/article/21/2/14-0623-
Techapp1.pdf). The isolates were identified as E. menin-
goseptica by using the Vitek II identification system (bio-
Mérieux, Marcy L’Étoile, France). However, MALDI-TOF
mass spectrometry identified strains HKU37 and HKU 38 as
E. meningoseptica (best match to E. meningoseptica strain
002_NEB14 NFI, with scores of 2.106 and 2.007, respec-
tively), whereas strain HKU36 was only identified to the
genus level as Elizabethkingia species (best match to E. me-
ningoseptica strain 002_NEB14 NFI, with score of 1.853)
(online Technical Appendix Figure 1). The isolates’ 16S
rRNA gene sequences exhibited 99.1%–99.9% nucleotide
identities to those of E. anophelis type strain R26T (Gen-
Bank accession no. EF426425) and 97.4%–99.9% nucleo-
tide identities to those of E. meningoseptica strains depos-
ited in GenBank (GenBank accession nos. HM056770.1,
GU180602.1, JQ673498.1, FJ816020, AVCQ01000012,
FJ839441.1, JN201943.1, and AJ704540).

234 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 2, February 2015
 E. anophelis Transmission from Mother to Infant

Figure 1. Clinical course of illness in 3 patients infected with Elizabethkingia anophelis in whom sepsis developed and the mother of
patient 1, who had culture-negative postpartum fever, Hong Kong, 2012. Locations where patients were treated at the hospital and times
when they were home are noted. CSF, cerebrospinal fluid; leaking, leaking of amniotic fluid (membrane rupture).

The high sequence identities to both E. anophelis and CBYE010000001–CBYE010000032, CBYF010000001–

E. meningoseptica made the species identity of the 3 strains
uncertain, despite 16S rRNA gene sequencing. Moreover,
the strains exhibited minor differences in phenotypes and
antibiogram (Table). Further, because the mothers stayed
in the same ward before delivery (although 4 months
apart), concerns of a possible nosocomial outbreak were
raised. However, environmental and water samples from
the hospital and patients’ homes were culture-negative for
E. anophelis. A program of enhanced infection control
measures was enforced in the hospital, and no further cases
were identified.
Genome Sequencing and Comparative Analysis
of E. anophelis Genomes
We sequenced the draft genomes of strains HKU36–38 to
investigate their genetic relatedness and confirm their spe-
cies identity. Sequencing generated 11–15 million paired-
end reads per strain (estimated 410–540-fold coverage).
After de novo assembly, the 3 draft genomes ranged from
3.92–3.99 Mb in length (G + C content 35.4%–35.8%)
and were distributed in 42–52 large (>500 bp) contigs
(EMBLaccessionnos.CBYD010000001–CBYD010000042,
CBYF010000038; online Technical Appendix Table 2).
These contigs contained 3,654–3,667 predicted protein-
coding genes (Figure 2, panel A). Using Genome-to-
Genome Distance Calculator for intergenomic distance
estimation, which enabled genome-based species delin-
eation analogous to traditional DNA–DNA hybridization
method, we found that these genomes shared 78.3%–
85.4% nucleotide identities to the draft genome sequence
of E. anophelis type strain R26T, the initial isolate from
an Anopheles gambiae mosquito (GenBank accession no.
NZ_ANIW00000000.1). However, the genomes shared
only 23.6%–23.7% nucleotide identities to the draft ge-
nome sequence of E. meningoseptica type strain ATCC
13253T (GenBank accession no. BARD00000000.1) (Fig-
ure 2, panel B). Phylogenetic analysis using the draft ge-
nomes and concatenated sequences of 69 housekeeping
genes also supported the identification of the 3 strains
as E. anophelis (Figure 3; online Technical Appendix
Figure 2).
The sequences from 52 contigs of strain HKU37 dem-
onstrated 99.4% nucleotide identity to those from 46 con-
tigs of strain HKU38, indicating that these draft genomes

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 2, February 2015 235
RESEARCH

Figure 2. Comparison of draft

genome sequence data of the
3 Elizabethkingia anophelis
strains from patients in Hong
Kong (HKU36–38), E anophelis
type strain R26T, and E.
meningoseptica type strain
ATCC 13253T. A) Distributions
of predicted coding sequence
function in genomes of E.
anophelis strains HKU36–38, E.
anophelis type strain R26T, and
E. meningoseptica type strain
ATCC 13253T according to SEED
Subsystems are shown. The
columns indicate the number of
proteins in different subsystems.
B) Circular representation of
sequence comparison between
the draft genome of strain HKU37
and other draft genomes as
labeled. Comparison generated
in Rapid Annotations using
Subsystem Technology (27).
Intensity of color indicates degree
of protein identity.

are essentially identical (Figure 2, panel B, and Figure 3).
The small intergenomic distance can be explained by slight
differences in coverage or contig assembly; sequences of
2,000 high-coverage protein-coding genes were identical
between HKU37 and HKU38. In contrast, these sequenc-
es demonstrated only 78.6% nucleotide identity to those
from the 42 contigs of strain HKU36, indicating that strain
HKU36 is genetically divergent (Figure 2, panel B, and
Figure 3), consistent with PFGE patterns (Figure 4). More-
over, a potential genetic island consisting of conjugative
transposable elements was found in strains HKU37 and
HKU38 but not in HKU36. Our results exclude a clonal
outbreak, but the extremely close genetic relatedness be-
tween strains HKU37 and HKU38 provides evidence for
vertical transmission from patient 2 to patient 3 (mother
to infant).

236 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 2, February 2015
 E. anophelis Transmission from Mother to Infant

Figure 3. Phylogenetic trees constructed by using draft genome sequences and concatenated sequences of 69 housekeeping genes of
3 Elizabethkingia anophelis strains from patients in Hong Kong (HKU36–38). A) Neighbor-joining tree constructed on the basis of draft
genome sequences using by using Genome-to-Genome Distance Calculator 2.0 (http://ggdc.dsmz.de/distcalc2.php; formula 1) and
Chryseobacterium gleum ATCC 35910 as the root. Arrow indicates route of mother-to-neonate transmission. B) Maximum-likelihood
tree constructed on the basis of 69 housekeeping genes, showing the relationship of E. anophelis strains HKU36–38 to related bacterial
species, using RAxML version 7.2.8 (http://sco.h-its.org/exelixis/software.html) and Weeksella virosa DSM 16922 as the root. A total
of 78,520 nt positions were included in the analysis. Bootstrap values were calculated from 1,000 replicates. Scale bars indicate mean
number of nucleotide substitutions per site on the respective branches. Gene names and accession numbers are given as cited in
GenBank (online Technical Appendix Table 2, http://wwwnc.cdc.gov/EID/article/21/2/14-0623-Techapp1.pdf). ‘E. meningoseptica’ strain
502 is a misidentified isolate that actually belongs to E. anophelis on the basis of draft genome sequencing.

Potential Virulence Factors and Resistance
Genes in E. anophelis
The association of E. anophelis with neonatal meningitis
in this and previous reports (17,18) suggests that the bac-
terium may possess virulence factors that enable it to in-
vade the central nervous system. The 3 draft genomes we
identified contain homologs of several virulence genes
found in Listeria monocytogenes, which also causes neo-
natal meningitis. These genes include cell wall hydrolase
A, which enables host cell invasion; phosphatidylino-
sitol-specific phospholipase (PlcA) and listeriolysin O
(LLO), which enable escape from the primary vacuole
of macrophages, and genes that enable survival in the
secondary vacuole of macrophages; and virulence clus-
ter protein B (VclB). Phosphatidylinositol-specific phos-
pholipase, listeriolysin O, and virulence cluster protein
B are located in the Listeria pathogenicity island LIPI-1
(30,31). Moreover, the 3 genomes we identified contain
homologs of arylsulfatase and genes that enable invasion
of brain endothelial cells, which contribute to the abil-
ity of Escherichia coli to cross the blood–brain barrier in
neonatal meningitis (32).
Vertical transmission of E. anophelis from mother to
infant also suggests that the bacterium may be able to colo-
nize the vagina before causing ascending chorioamnionitis
in the mother and neonatal infection through transplacental
spread. A homologof the gene encoding agmatine deimi-
nase, AgDI, which mediates acid tolerance in L. mono-
cytogenes (33), was found in the E. anophelis genomes.
Further studies may investigate the possible role of AgDI
and potential adherence factors for vaginal colonization in
E. anophelis.
Similar to E. meningoseptica, the 3 E. anophelis iso-
lates we identified are resistant to multiple antimicrobial
drugs. We found various antimicrobial resistance genes
consistent with their resistance phenotypes, including
metallo-β-lactamase (blaGOB-1 and blaB14 in strain HKU36
and a novel blaGOB and blaB1 in strains HKU37 and HKU38)
and extended-spectrum β-lactamase (blaACME-1 in strains
HKU37 and HKU38 and a potential novel blaACME-1 vari-
ant in strain HKU36). A comparison of these β-lactamases
to their corresponding orthologs in E. meningoseptica ge-
nomes revealed only 74%–85% amino acid identities, in-
dicating that E. anophelis and related bacteria are potential
reservoirs of novel β-lactamase genes (19,34,35). Other
antimicrobial resistance genes found included multidrug-
resistance efflux pumps (ATP binding cassette superfam-
ily, major facilitator superfamily, resistance-nodulation-
division families, multidrug and toxic-compound extrusion
family) that potentially carry resistance to a variety of
compounds; chloramphenicol acetyltransferase; amino-
glycoside 6-adenyltransferase; and tetracycline resistant
gene. Moreover, a putative tetX gene was also identified;
this gene encodes a predicted flavin-dependent monooxy-
genase with tetracycline/tigecycline-degrading activity,
although the 3 strains we identified are only resistant to tet-
racycline but remained susceptible to other related drugs,
including tigecycline.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 2, February 2015 237
RESEARCH
Comparison of Genomes from Human and
Mosquito E. anophelis Strains
E. anophelis strains R26T and Ag1 were isolated from mos-
quitoes (35). Compared with those strains, the genomes of
the 3 strains we identified possessed 33 unique hypothetical
proteins. Moreover, the genetic island consisting of conju-
gative transposable elements found in strains HKU37 and
HKU38 was also absent in the mosquito strains. In contrast
to the mosquito strains, which possessed genes encoding
for xylose isomerase (XylA) and xylulose kinase (XylB),
these 2 genes were absent in the 3 strains we identified.
This finding may reflect different requirements for sugar
metabolism in E. anophelis under different environments.
Notably, despite the presence of XylA and XylB, E. anoph-
elis mosquito strain R26T did not produce acid from xylose
(15). However, this finding does not exclude the strain’s
ability to metabolize xylose, as D-xylulose 5-phosphate,
the product of XylA and XylB, can be used as a substrate
for the pentose-phosphate pathway. XylA and XylB were
238 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 2, February 2015
Figure 4. Pulsed-field gel
electrophoresis (PFGE) analysis of
samples from patients in Hong Kong
showing 3 Elizabethkingia anophelis
strains compared with reference
Elizabethkingia isolates. A) PFGE
performed by using CHEF Mapper XA
system (Bio-Rad, Hercules, CA, USA)
and restriction endonuclease XbaI
shows that isolates from patient 2 and
patient 3 are indistinguishable, wheras
isolates from patient 1 possess distinct
PFGE patterns. Lane 1, E. anophelis
strain HKU37 from uterine swab
specimen of patient 2; lane 2, placental
swab specimen from patient 2; lane 3,
E. anophelis strain HKU38 from blood
of patient 3; lane 4, cerebrospinal fluid
from patient 3; lane 5, E. anophelis
strain HKU36 from blood of patient 1;
lane 6, cerebrospinal fluid from patient
1; lane 7, E. anophelis type strain
R26T; lane 8, E. meningoseptica type
strain ATCC 13253T; lane 9,
E. miricola type strain LMG22470T.
B) Dendrogram constructed with
PFGE data by similarity and clustering
analysis using the Dice coefficient
(1% tolerance and 0.5% optimization)
and the unweighted pair-group
method using average linkages with
GelCompar II (Applied Maths,
Sint-Martens-Latem, Belgium).
also absent in the genome of E. meningoseptica type strain
ATCC 13253T, which suggests that mosquito strains of
E. anophelis may be evolutionarily distinct from clinical
strains of E. anophelis and E. meningoseptica. More ge-
nome sequence data from other clinical and environmental
strains of E. anophelis may shed light on the ecology, biol-
ogy, and pathogenesis of E. anophelis.
Discussion
This study demonstrates the power of draft genome se-
quencing to rapidly dissect transmission pathways for
emerging bacterial infections. Our results showed that ver-
tical perinatal transmission had occurred from patient 2, a
pregnant woman who had chorioamnionitis, to patient 3, a
neonate who had early onset neonatal meningitis. The in-
fective source for patients 2 and 3 was unlikely to have
been patient 1 or his mother. However, we speculate that
the mother of patient 1 might also have had E. anophelis
chorioamnionitis, as evidenced by postpartum fever and

abdominal pain, which resulted in late-onset meningitis
in her son owing to fastidious bacterial growth. Although
strain HKU36 did not belong to the same clone as strains
HKU37/38, a polyclonal outbreak of E. anophelis sepsis in
the labor ward, in which case an environmental source is
likely, could not be excluded.
The discovery of E. anophelis in mosquito gut has
raised suspicion that mosquitoes are the source of neonatal
meningitis cases in Africa (17). Although Anopheles mos-
quitoes are not found in Hong Kong, the role of local mos-
quitoes as reservoirs for E. anophelis remains unknown.
Nonetheless, the vertical transmission demonstrated in 1
neonate makes mosquitoes unlikely as vehicles of trans-
mission in our cases.
Our report provides genomic evidence for vertical
transmission in neonatal meningitis. Whereas we cannot
ascertain how the mother(s) acquired the infection, our re-
sults prompt further work to assess the importance of ma-
ternal source in neonatal meningitis caused by E. anoph-
elis and other bacterial agents. Maternal colonization with
Lancefield group B streptococcus (GBS) during pregnancy
is the primary risk factor for early onset neonatal disease.
However, direct microbiological evidence for vertical
transmission is seldom available, especially for bacterial
agents other than GBS. Further genomic studies may help
investigate the role of vertical transmission in neonatal
meningitis caused by other bacteria. Current indications
for intrapartum antimicrobial drugs prophylaxis have been
determined on the basis of risk factors for early onset GBS
disease; therefore, intravenous penicillin G or ampicillin
is often the standard empirical regimen used. However, if
further research determines that the mother may also be a
source of transmission for other bacterial agents, broader-
spectrum antimicrobial drugs may need to be considered
as treatment for intrapartum fever or prolonged rupture
of membranes.
E. anophelis is likely an underreported bacterium be-
cause it can be easily misidentified as E. meningoseptica,
which shares a similar phenotypic profile (17,19). The E.
anophelis isolates from the recent outbreak reported in
Singapore were initially mistakenly identified as E. menin-
goseptica (19,36). Of the 3 strains we identified, 2 were
misidentified as E. meningoseptica with MALDI-TOF
mass spectrometry, the state-of-the-art technology, which
is replacing conventional phenotypic identification in diag-
nostic laboratories. The reason for failure of MALDI-TOF
mass spectrometry to identify these strains was that refer-
ence E. anophelis strains are lacking in existing diagnostic
spectrum databases, as is the case with other less common-
ly encountered organisms (21).
Although 16S rRNA gene sequencing should provide
sufficient resolution, some strains indexed as E. meningo-
septica, such as strains G3-1-08 and 502, were actually
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 21, No. 2, February 2015 239
E. anophelis Transmission from Mother to Infant
more closely related to E. anophelis than to E. meningo-
septica in their 16S rRNA sequences (Figure 3; online
Technical Appendix Figure 2) (37). These ambiguous, po-
tentially misidentified strains may cause incorrect interpre-
tations in suspected E. anophelis infections. For example,
the sequence of strain HKU36 possessed 99.8% nucleotide
identity to that of E. meningingoseptica strain G3-1-08 but
only 99.1% nucleotide identity to that of E. anophelis strain
R26T. Furthermore, phenotypic tests such as acid produc-
tion from cellobiose and citrate utilization, previously pro-
posed to be useful for identification of E. anophelis (15),
are probably unreliable in differentiating among Elizabeth-
kingia species. For example, E. anophelis strain R26T pro-
duces acid from cellobiose, but the 3 strains we identified
do not; in addition, E. anophelis strains R26T, HKU37, and
HKU38, but not strain HKU36, utilize citrate (online Tech-
nical Appendix Table 1). Strain HKU36 displayed higher
MIC of vancomycin than did strains HKU37 and HKU38
and type strains of E. anophelis, E. meningoseptica, and
E. miricola, which correlates with previous reports on
variable vancomycin susceptibilities in Elizabethkingia
(38,39). The species identity of the 3 strains we identified
was only resolved by intergenomic comparison. Inclusion
of E. anophelis in MALDI-TOF MS databases and recti-
fication of 16S rRNA gene sequences of Elizabethkingia
strains deposited in databases will enable accurate diagno-
sis of more E. anophelis infections.
The draft genome sequences we identified have en-
abled rapid exploration of novel β-lactamase and other
antimicrobial drug resistance genes and possible viru-
lence genes in E. anophelis, highlighting the potential of
genome sequencing in identifying novel drug-resistance
mechanisms and guiding treatment regimens for emerging,
multidrug-resistant bacteria (25,34,40). Because previous
cases of E. anophelis neonatal meningitis have been associ-
ated with poor outcomes (17,18), further work to elucidate
the pathogenesis and antimicrobial drug resistance patterns
of this emerging pathogen may help improve clinical man-
agement of illness. The findings of potential genes related
to neuroinvasion and acid tolerance and the unique genetic
characteristics in clinical strains of E. anophelis compared
with mosquito strains may also provide insights on the abil-
ity of E. anophelis to adapt to different ecologic niches and
cause neonatal infection through vertical transmission.
In conclusion, the genome data we obtained for these
cases offered superior discriminatory power that supported
appropriate infection control measures. The ability to dis-
tinguish different bacterial isolates often has critical im-
plications on practical infection-control management, but
different strains of the same bacterial species may not be
distinguishable by their phenotypes because they reflect
a tiny portion of the microbial genome. With better auto-
mation and lower costs, draft genome sequencing, which
RESEARCH
offers a short turnaround time, may replace existing typing
methods such as PFGE or multilocus sequence typing for
outbreak investigations.
Acknowledgments
We thank Cheung-Hing Foo for technical support in bacterial
identification and members of the Centre for Genomic Sciences,
The University of Hong Kong, for their technical support in ge-
nome sequencing.
This work was supported by Health and Medical Research Fund,
Food and Health Bureau, The Government of the Hong Kong
Special Administrative Region; Strategic Research Theme Fund,
Committee for Research and Conference Grant, and University
Development Fund, The University of Hong Kong; the Shaw
Foundation; donation from Ms. Eunice Lam; and Consultancy
Service for Enhancing Laboratory Surveillance of Emerging In-
fectious Disease for the HKSAR Department of Health.
Dr. Lau is a clinical professor in the Department of Microbiology
at The University of Hong Kong. Her research focuses on micro-
bial genomics for studying emerging infectious diseases.
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Address for correspondence: Patrick C.Y. Woo, Department of
Microbiology, The University of Hong Kong, Room 423, University
Pathology Building, Queen Mary Hospital Compound, Pokfulam Road,
Hong Kong, China; email: pcywoo@hkucc.hku.hk
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